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Structure/function analysis of CD40; a key activator of B lymphocytes

University of British Columbia

B lymphocytes need two signals in order to differentiate into antibody-producing cells, one delivered by the B cell antigen receptor (BCR) and a second delivered by CD40. In the absence of the CD40 signal, B cells that receive only the BCR signal are rendered non-responsive or undergo apoptosis The ability of CD40 to rescue B cells from BCR-induced apoptosis can be demonstrated using the WEHI-231 B lymphoma cell line. I have used this cell line to investigate the role of mitogen-activated protein (MAP) kinases in integrating BCR and CD40 signaling. The three types of MAP kinases, the ERKs, the c-Jun N-terminal kinases (JNKs), and p38, each phosphorylate a distinct set of transcription factors. Thus, activating different combinations of MAP kinases could lead to distinct biological responses. I found that BCR engagement in WEHI-231 cells strongly activates ERK2 and weakly activates ERK1, JNK and p38. CD40 engagement did not activate either of these kinases, nor did it affect BCR-induced ERK activation. In contrast, CD40 engagement markedly stimulates JNK and p38 as well as MAPKAP kinase-2, a downstream target of p38. The BCR weakly activates JNK and p38 by itself, however, it potentiates CD40-induced JNK activation. Thus, activation of ERK2 alone correlates with apoptosis in WEHI-231 cells, whereas full activation of all three MAP kinase pathways correlates with cell survival. The role of MAP kinases in regulating these responses remains to be tested. To identify signaling motifs in the CD40 cytoplasmic domain that are responsible for activation of the JNK and p38 MAP kinases, I created a set of 12 chimeric receptors consisting of the extracellular and transmembrane domains of CD8 fused to portions of the murine CD40 cytoplasmic domain. These chimeric receptors were expressed in WEHI-231 B lymphoma cells. I found that amino acids 35-45 of the CD40 cytoplasmic domain constitute an independent signaling motif that is sufficient for activation of the JNK and p38 MAP kinase pathways, as well as for induction of IκBα phosphorylation and degradation. Amino acids 35-45 were also sufficient to protect WEHI-231 cells from anti-lgM-induced growth arrest. This is the same region of CD40 required for binding to the TRAF2, TRAF3 and TRAF5 adapter proteins. These data support the idea that one or more of these TRAF proteins couple CD40 to the kinase cascades that activate NF-κB , JNK and p38. Another aim of this thesis was to test the hypothesis that ATAR, a recently discovered tumor necrosis factor (TNF) superfamily receptor, can mimic the effects of CD40 on B cells. Like CD40, ATAR is expressed on B cells and interacts with ligands expressed by activated T cells. To study ATAR signaling, two chimeric receptors consisting of the extracellular and transmembrane domains of CD8 fused to portions of the ATAR cytoplasmic domain were constructed and expressed in WEHI-231 cells. We found that the cytoplasmic tail of ATAR mediated phosphorylation of JNK and p38, phosphorylation and degradation of IκBα, as well as protection of WEHI-231 cells from anti-lgM-induced growth arrest. The C-terminal portion of the ATAR tail containing the TRAF-interaction domain was sufficient to mediate these signaling events. Our results support a model in which TRAF2 and/or TRAF5 link ATAR to the activation of JNK, p38 and NF-κB, as well as to B cell survival. The ability of ATAR to mimic some of the effects of CD40 on B cells suggests that this novel TNFR superfamily member may provide an alternative second signal to B cells. In addition to B lymphocytes, CD40 is highly expressed on dendritic cells (DC). Both CD40 and lipopolysaccharide (LPS) have been shown to activate these antigen presenting cells. The final aim of this thesis was to determine whether the MAP kinases might be involved in CD40 or LPS-induced activation of DCs. I tested whether ERK, JNK or p38 are activated by CD40 and LPS in the murine D1 DC line. I found that both CD40 and LPS strongly activated ERK2 in D1 cells. In contrast, little or no activation of JNK and MAPKAP kinase-2 was induced by either of these stimuli. Our collaborators extended these findings by showing that ERK activation is essential for the ability of LPS to protect DCs from growth factor withdrawal-induced apoptosis.

Thesis/Dissertation

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2009-07-03

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http://hdl.handle.net/2429/10033

Microbiology and Immunology

University of British Columbia

UBCV

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